Many studies and developments have been conducted for a wide range of applications of so-called biosensors and bioreactors that utilize molecular recognition ability and substance-converting ability of biological substances such as enzymes and antibodies as well as nucleic acid molecules (e.g., DNA and RNA) including genes.
For the biosensors, there are growing demands on further technical developments for applications on various detection targets in conjunction with growing interests in health as well as matters of environmental pollutants and public safety thereof. Recently, furthermore, the bioreactors have attracted much attention as eco-friendly clean processing technologies. Therefore, for example, there are increasing demands on further technical developments such as those in processes of producing products utilizing various bioprocesses.
For the biosensors, specifically, detectors for detecting objective ones by utilizing the selective molecular recognition of respective biological substance molecules have been developed extensively. For example, detectors developed on the basis of various kinds of detection procedures include a DNA sensor chip that utilizes a base-sequence-dependent complimentary hydrogen bonding between deoxyribonucleic acid (hereinafter, referred to as DNA) sequences (i.e., a hybridization reaction between the complimentary strands), an antibody sensor that detects a disease marker or the like to be eluted in blood, by utilizing a molecular recognition ability, originated from a specific binding ability between a protein molecule and a low-molecular substance or between protein molecules such as an antigen-antibody reaction, and an enzyme sensor for detecting the level of a substrate substance by utilizing an oxidation-reduction enzyme or a hydrolytic enzyme, as typified by a glucose sensor for a diabetic patient.
Currently, the biosensor that makes use of any of these biological substances, generally employs a system of using, in the form of a biological substance-immobilized substrate, a biological substance to be used, for example a nucleic acid molecule such as DNA, or proteins of antibodies, enzymes, etc., which is immobilized on the surface of a substrate such as a flat plate, a sphere or a materials, or the like.
In addition, one of the performance qualities required for the biosensors being developed nowadays is “high sensitivity and downsizing”, which is typified by μ-TAS. For attaining an object of “high sensitivity and downsizing”, an important technical issue is how to effectively utilize a minute space of a reaction field or detection field and how to increase the sensitivity of the biosensor.
For instance, in the detection field where the biological substance is immobilized on the substrate, in addition to the specific binding to a target substance to be detected, there is a possibility of causing much non-specific adsorption of biological substances except the substance to be detected or a possibility of causing a non-specific binding of the substance to be detected itself on the substrate. These non-specific adsorbing phenomena will become one of the factors that decrease a Signal/Noise ratio of the biosensor. In particular, the total amount of the specific binding of the target substance to be detected falls off as the detection field decreases. Therefore, the biosensor tends to be influenced by noises due to the non-specific adsorption, resulting in difficulty in high sensitive measurement. Also, in terms of an effective utilization of a sample in minute amounts, it is difficult to carry out measurement at a sufficiently high accuracy when the non-specific adsorption of the target substance to be detected is caused in large quantities. Hereafter, therefore, an important technical problem which remains to be improved is to reduce or prevent the non-specific adsorption phenomenon.
On the other hand, for the bioreactors, there have developed procedures for producing food additives such as amino acids, candidate substances for medicines and antibiotics by enzymatic reactions that mainly employ the position-selective catalytic functions of enzymes as one type of proteins instead of procedures that utilize microorganisms having the abilities of producing objective products. Besides, the applications of enzymatic reactions to the productions of chemicals and polymer materials have been also under study. In the development of bioreactors using such enzymatic reactions, because the development of devices suitable for high-mix low-volume production has been also mainstream, for example, with the spread of a technique for screening a candidate substance by means of combinatorial chemistry, there are increasing demands for miniaturizing individual biosensors by means of a device on which an enzyme to be used in a reaction just as in the case with the biosensor described above is immobilized (i.e., for high-mix low-volume production).
In addition, materials, which can be employed for substrates, flat plates, spheres and porous materials, or the like, for biological substance-immobilized substrates to be used in the biosensors and bioreactors, generally include organic polymers, glass, ceramics, metal flat plates, and other materials known in the art, depending on the types and applications of the biological substances.
As a method of immobilizing a biological substance such as protein, on the surface of substrate, for example, there is an immobilizing procedure using physical adsorption, which includes the steps of forming a coating layer of a protein solution on the surface of a substrate by using means for dipping the substrate into the protein solution or applying the protein solution thereon, and then removing a solvent from the coating layer and drying it to allow the protein to be immobilized on the surface of the substrate as a result of physical adsorption. Alternatively, there is another procedure that includes next two steps, the first step is chemically modifying the surfaces of a substrate or the protein molecules to provide the high activity functional groups, and second step is immobilization of the protein molecules on the surface of the substrate through the chemical bonding by forming of chemical bonding between the introduced the high activity functional groups and other functional groups. These procedures have been hitherto known as those for immobilizing biological substances on the surfaces of substrate. As an example of the immobilizing method using physical adsorption, JP 06-003317 A discloses a method of manufacturing an enzymatic electrode by the application of a method including the steps of forming a charge-transporting organic complex layer on the surface of a conductive substrate and then applying a protein solution on the charge-transporting organic complex layer, followed by drying the protein layer to allow an enzyme protein to be physically adsorbed and immobilized on the surface of the substrate through the charge-transporting organic complex layer.
As an example of the immobilizing method using the chemical bonding, Sensor and Actuators B15-16 p 127 (1993) discloses a method including the steps of subjecting a platinum-deposited surface of a silicon substrate to treatment with an amine-based silane coupling agent and then coupling between an amino group derived from the amino-silane coupling and a peptide chain by means of a cross-linking agent such as glutaric aldehyde to carry out immobilization. In addition, for making a detector such as a biosensor-composed of antibodies immobilized on a glass substrate, a method is applied, in which reactive functional groups are introduced to the surface of the glass substrate by means of treatment with a silane coupling agent and a peptide chain is immobilized through a chemical bonding using a cross-lining agent as described above.
However, in the immobilizing method based on physical adsorption or the chemically-immobilizing method based on chemical cross-linking, the portion of a protein, which is used for adsorption or binding to the substrate can be selected at random. Therefore, when a portion, which directly or indirectly relates to the binding ability required for the protein, the enzymatic activity of the protein, or the like, also becomes one relating to the binding to the surface of a substrate, there is a fear that a desired function of the protein will deteriorate remarkably if the protein binds to the substrate.
Therefore, it becomes important to develop means for previously determining an immobilizing portion of a molecule to be immobilized, which will be used for binding to the surface of the substrate, for example, a technology capable of previously controlling the orientation of a biological substance to be immobilized on the surface of the substrate.
Furthermore, for attaining “high sensitivity and downsizing”, the biological substance should be integrated very densely in a very small area on the surface of the substrate and then immobilized thereon.
As an example of a method of integrating the biological substance very densely and immobilizing the same, there is a method well known in the art, where a substrate having a large specific surface area, for example a porous material having a regular nanoporous structure, is adopted as a substrate, and a biological substance is then immobilized on the surface having a porous structure with a large specific surface area. As a conventional method for forming the porous structure having regularity with a scale in the order of nanometers, which can be used for the above purpose, a polymer membrane filter, porous glass, anodized aluminum oxide film, and so on are well known in the art. For the anodized aluminum oxide film, in particular, the pore size thereof can be regulated by means of a voltage applied at the time of oxidation to make a film having a given pore size in the order of nanometers.
Making the porous material into the substrate enables a reaction field on which a biological substance is immobilized in an amount enough for high-sensitivity detection even in a very small area.
Conventional examples of the method using the porous substrate described above as a substrate, particularly the method by which a biological substance such as a protein is immobilized on an anodized aluminum oxide film, include the following procedures:
As an example of a procedure for covalently binding a protein using a cross-linking agent after surface treatment with an amino-silane coupling agent, U.S. Pat. No. 6,225,131 discloses a method including the steps of providing a commercially-available aluminum oxide film as a substrate, subjecting the surface thereof to treatment with 3-aminopropyltriethoxysilane (APS), and covalently binding anti-human chorionic gonadotropin mouse monoclonal antibodies using glutaric aldehyde as a cross-linking agent to immobilize them on the surface of the substrate.
Furthermore, as an example of a procedure using intermolecular binding between an organic substance and a peptide, US 2002/0106702 A1 discloses a method by which an organic thin film for binding a protein is arranged on an aluminum oxide film to immobilize a protein fused with a peptide chain having affinity to an organic substrate that constitutes an organic thin film described above.
The above substrate having a large specific surface area, such as a porous material having a regular nanoporous structure, is adopted as a substrate to allow a larger amount of the biological substance to be immobilized on the surface of the substrate. However, when the biological substance immobilized on the substrate does not take an orientation suitable for the binding to a target substance to be detected, the detection sensitivity corresponding to the amount of the biological substance immobilized may not be attained. Also, when a biological substance does not have an orientation suitable for the substrate substance on which the biological substance acts, the reactivity corresponding to an amount of the biological substance to be immobilized is not attained in some cases. That is, unless a biological substance to be immobilized on a substrate is immobilized after controlling the orientation suitable for the use thereof, the biological substance will be insufficient to exert its advantage accompanying immobilization of a larger amount of the biological substance on the surface of the substrate through the use of a substrate having a large specific surface area.
In other words, unless a biological substance to be immobilized on a substrate is immobilized after controlling the orientation suitable for the use thereof, it becomes necessary to further increase the amount of the biological substance to be immobilized on the substrate to obtain the desired detection sensitivity or reactivity. Thus, there is a possibility that an excess amount of the biological substance per unit area of the substrate should be immobilized or the area of the substrate on which the biological substance is immobilized should be excessively extended. When the area of the substrate on which the biological substance is immobilized is extended excessively, it may become a large obstacle to the downsizing of a device itself.
Furthermore, in the case of ingredients in the biological substance, which will cost high upon their preparation, there is a possibility of increasing the total cost of the device when they will be used in large amounts. Furthermore, a procedure of forming an additional adhesion layer for binding an organic substance to a substrate (i.e., a layer formed between the substrate and the organic substance and having a configuration different from that of the organic substance to be immobilized) may involve an increase in the number of steps required and become a large obstacle to a decrease in device cost.
In addition, a high technical level is also required for completely forming the adhesion layer on the inner-wall surface of the porous portion of the nanoporous structure. If the formation of the adhesion layer in the inside of the pore is insufficient, an effect obtained by increasing the specific surface area by means of the porous structure may be insufficiently exerted.
In view of the present situation, such a problem cannot be coped with any publicly known technology of immobilizing ingredients in the biological substance by means of chemical bonding between the ingredients and the substrate with physical adsorption through the adhesion layer or non-specific modification using a cross-linking agent.
Therefore, it has been desired to provide a structure composed of a substrate and an organic substance immobilized on the surface of the substrate such that the molecular orientation of the organic substance is regulated so as to exert its desired functions, and a concise immobilizing procedure that allows the organic substance to be immobilized on the surface of the substrate.